Removal of microorganisms from fluids



United States Patent 3,242,073 REMOVAL OF MICROORGANISMS FROM FLUIDSKenneth W. Guebert and Johnnie D. Laman, Lake Jackson, Tex., assignorsto The Dow Chemical Company, Midland, Mich., a corporation of DelawareNo Drawing. Filed Mar. 7, 1963, Ser. No. 263,417

9 Claims. (Cl. 210-64) The present invention concerns the removal ofmicroorganisms from fluids. More specifically, the invention concerns arapid flow rate filtration method capable of separating microorganismsfrom large volumes of fluids.

A wide variety of filters are presently employed in methods designed toeffect removal of microorganisms from fluids. Examples of these filtersinclude porous sintered glass, nitro-cellulose ester, porous porcelainand asbestos media. These methods generally depend upon mechanicalentrapment to achieve their purpose and this dependance necessitates theuse of filter media which have openings or passageways of a magnitudecomparable to that of the microorganisms to be removed. The extremelysmall passageways required are responsible for a high pressure dropbetween the influent and efl'luent sides of the filter and a slow rateof flow during fluid filtration. Other inherent disadvantages of thefilters employed in the prior art methods include low capacities, due torapid clogging of the minute passageways, and high cost. In addition,the microorganisms filtered from the fluid frequently have rapid growthrates and spread through the filter media to the efiluent side wherecontamination of the filtrate occurs. These filter media must usually bepre-sterilized and carefully protected against contamination prior touse. Although some of these known filter media are capable of completelyremoving bacteria from small quantities of liquids, they are generallyineffective in removing smaller sized microorganisms such as viruses.

It is an object of the present invention to provide a method for theremoval of microorganisms from fluids by filtration. It is anotherobject to provide such a method characterized by rapid flow rates andlong filter cycles. Another object is to provide such a method employingfilter media which are unaffected by exposure to nonsterile conditionsprior to use. It is another object to provide such a method whichremoves both bacterial and viral microorganisms from fluids. Furtherobjects and advantages of the present invention will be evident from thefollowing description.

In the method of the present invention microorganisms are removed fromfluids by passing the fluids through a specially prepared filter mediumwhich comprises a conventional anionic-type filter aid having acationic, or ganic, polyelectrolyte coating. The term filter aid will(be used herein to designate any one or more of the wide variety offibrous or particulate materials employed for filtration purposes. Alarge majority of filter aids are characterized by an electronegativelycharged surface and these will be referred to as anionic-type filteraids. Examples of anionic-type filter aids, which are suitable for usein preparing the coated filter media employed in the method of thepresent invention, include diatomaceous earth, paper filter pulp,fullers earth, charcoal, anthracite coal, sand and the like.

The use of the specially prepared filter media described above enablesthe removal of microorganisms from large volumes of fluids passedthrough the coated filter aid media at rapid flow rates. These mediashow tenacious retention of the microorganisms, thus obviating thenecessity for pre-sterilization and elaborate handling precautions,prior to use, and reducing the previously discussed problem of filtratecontamination resulting from the normally rapid growth of microorganismsthrough a filter to the effluent side.

The coated filter aids employed in the method of the present inventionare prepared by treating an anionictype filter aid with a small amountof a cationic, organic, {polyelectrolyte This treatment isadvantageously accomplished by spraying the filter aid with a dilutesolution of the cationic polyelectrolyte, or by addition of the polyelectrolyte to a slurry of the filter aid followed by drying of theresulting coated filter aid at a temperature less than the decompositiontemperature of the polyelectrolyte coating on the filter aid.Alternatively, the cationic polyelectrolyte may be added to a slurry ofthe filter aid prior to deposition of the filter aid on the filtersupport.

The organic, cationic, polyelectrolytes employed in preparing .thecoated filter aids used in the method of the present invention form anadherent surface coating on the anionic-type filter aids due to theelectrostatic attraction of the oppositely charged materials. It isessential that only a portion of the total cationic charge sites of thepolyelectrolyte is neutralized in this bonding action, thus leavingpositively charged sites available to attract and hold microorganismswhich are to be removed from fluids passed through the coated filter aidmaterial. In order to provide these non-neutralized .sites, thepolyelectrolyte must be substantially linear and must contain a minimumnumber of available charge sites per molecule. This minimum number isabout ten charge sites per molecule, but a substantially larger numberof charge sites per molecule is preferred. Cationic polyelectrolyteswhich are especially efiicacious for use in the treatment ofanionic-type filter aids, for use in the method of the presentinvention, are those prepared (1) by the polymerization ofalkylenimines, e.g. ethylenimine, to form polyalkylenimines and (2) bythe condensation reaction of :dihaloalkanes with polyalkylenepolyamines,e.g. ethylene dichloride and triethylenetetraamine, to form polymericpolyalkylenepolyamines. These polymers possess a very high ratio ofcationic charge sites to molecular weight and are conveniently employedin aqueous solutions of the desired concentration, as explained below,to coat the anionic-type filter aids. Although these polymers arenormally water-soluble, they form an adherent coating on the filter aidwhich is not removed by contact with water. The molecular weight of thecationic polyelectrolyte is not critical provided that the previouslydis cussed minimum number of cationic charge sites are available.

Other examples of suitable cationic, organic, polyelectrolytes includepolyvinylbenzyltrimethyl ammonium chloride, dimethylaminoethylpolymethacrylate and copolymers of N-Z-hydroxyethyl aziridine and bis(3aziridinyl-Z-hyd roXy-n-propyl ether.

In summary, any substantially linear, cationic, organic polyelectrolytecontaining a minimum of about 10 available charge sites per molecule maybe employed to prepare coated filter aids for use in the method of thisinvention, although those polyelectrolytes having cationic groups whichare at least as basic as primary amine groups are most advantageouslyutilized. Those polyelectrolytes having the most strongly cationicgroups such as the onium groups, e.g., ammonium, phosphonium, pyridiniumand sulfonium groups, are preferred.

A concentration of from about 5 to 30 weight percent of the cationicpolyelectrolyte in aqueous solution is preferably employed as a stocksolution to spray-treat the anionic-type filter aid. The more dilutesolutions have a lower viscosity and are easier to use but result in theaddition of more water to the filter aid. The water does not interferewith the performance of the coated filter aid, but the added weight dueto water retained in the coated filter aid may be undesirable. When afilter aid slurry treatment procedure is employed the polymerconcentration in the slurry is not critical. Many particulate types offilter aids, such as diatomaceous earth, require large amounts of waterto form a slurry. In their case, a solution containing 0.2 percent byweight or less of polymer is advantageous. More concentrated solutionsmay result in an excess of polymer being added merely to obtainsufiicient liquid to obtain a slurry. This excess polymer will be lostwhen washing the coated filter aid, or during the filter cycle if thecoated material is not washed, and therefore increases costs or resultsin filtrate contamination. Irrespective of the filter aid coatingprocedure employed, more polyelectrolyte may be present than can be heldon the filter media by electrostatic attraction. This excesspolyelectrolyte will be washed off during use or when forming the coatedfilter media on the filter support. If the presence of this excesspolyelectrolyte is objectionable, the first volume of filtrate may bediverted or, alternatively, the coated filter aid may be washed beforeapplying onto the filter support.

Polymer concentrations less than 0.2 weight percent, e.g. in the rangeof 0.01 weight percent, may be used in the slurry if desired. Even moredilute solutions can be employed but this requires handling largeamounts of liquids in relation to the weight of filter aid coated sincea polymer coating of from about 0.1 to 0.2 weight percent, based on thecoated filter aid Weight, is generally preferred.

The treatment of anionic-type filter aids with these aqueous polymersolutions results in a filter aid material having from about 0.01 to2.00 weight percent polymer, based on total Weight of the coated filteraid, as an adherent surface coating. The amount of polymer coating willdepend upon the concentration of polymer in the treating solution, theconcentration of polymer cationic charge and the filter aid employed.

Although the method of the present invention is capable of handlinglarge volumes of fluids during extended filter cycles it should be notedthat the filter cycle length may be extended even further by theaddition of body feed, i.e. addition of untreated filter aid to theinfiuent fluid stream, such as is practiced in other types of knownfiltration procedures. The filter cycle may also be extended by theaddition of more of the cationic, organic, polyelectrolyte coatingmaterial to the coated filter aid when an appreciable percentage of thecationic charge sites originally available on the coated filter aid havecollected microorganisms during the filtration process. This addition,during the filter cycle, will re-coat the filter media and thusreplenish the cationic sites available to attract and holdmicroorganisms.

The following examples describe completely representative specificembodiments of the present invention. These examples are not to beinterpreted as limiting the invention other than as defined in theclaims.

EXAMPLE 1 The necessity of having a minimum of approximately availablecationic charge sites per molecule of polyelectrolyte used to coat theanionic-type filter aids is shown by the results in Table 1, below,These results were obtained by the following procedure.

A slurry was prepared by mixing 40 grams of diatomaceous earth,hereafter DE, and 100 ml. of an aqueous solution containing 0.2 weightpercent of the polyelectrolyte designated in Table I, below. This slurrywas gently stirred for minutes and deposited on a Buechner funnel,containing a filter paper to furnish support for the coated filter aid,to form a filter bed. This filter bed was then washed with four portionsof de-ionized water in order to remove any excess polymer. A quantity of500 ml. of water contaminated with bacteria was then passed through thefilter bed by gravity flow and bacteria counts determined for theeffluent.

1 Number of available cationic charge sites per molecule. 2Polyalkylencpolyainiuea condensation product of pentaethylenehexaarnineand ethylene dichloride having an average molecular weight 3Polyethylenimine-molecular weight range of 2,000 to 5,000.

EXAMPLE 2 A variety of bacteria were employed to contaminate sterilewater samples and these samples were then passed through various filtersto test their capabilities to remove these microorganisms. Twenty-fourhour broth cultures of Escherichia co li, Staphylococcus albus andaureus, alpha and gamma hemolytic Streptococcus and Aerobacter aerogeneswere employed as the microorganisms in these tests. One ml. of a testculture was added to mi. of sterile distilled water and total platecounts of the number of organisms present were determined by preparingserial dilutions.

The seeded samples were then filtered rapidly, under vacuum, throughsterile Buechner funnels containing a filter bed of DE coated with (1)an organic, cationic polyelectrolyte or (2) aluminum hydroxide. Anuntreated DE filter bed was used as a control. The polyethylenimine(hereafter PEI) coated DE was prepared by spraying the polymer as anapproximately 15 weight percent aqueous solution onto the DE. The otherpolyelectrolyte coatings were applied by the previosuly described slurrymethod. The aluminum hydroxide coated filter aid was prepared by formingan aqueous slurry of DE, aluminum sulfate and soda ash, agitating theslurry for 15 minutes, depositing the slurry on the filter support andwashing with water to remove excess materials. The coated filter aidcontained about 2 percent by weight of aluminum hydroxide. The bacterialcomparison counts of the influent and efiluent are recorded in Table 11,below.

Table I1 Bacteria count per ml.

DE filter aid Microorganism Pre-fil- Post-filtration tration Untreated210, 000 0 a Streptococcus 5, 000 0 0.2% PEL Aerobacter acr0gmes 490,000 0 1.0% polyazetidinc Acmbacter aerogmes; 50,000 0 1.0% polyaziridine2 Aerobuctcr aerogmes. 82, 000 0 D0. Gamma Streptococcus 23, 000 0 Do 2E. coli 62,000 0 1 Polyazetidinepolymeric N,N-diethyl-3-hydroxyazetidiue. Z Polyazir1(linecopolymer of N-Z-hydroxyethyl aziridine andbis(3- aziridinyl-2-hydroxy-n-propyl) ether.

Although the inorganic coated DE produced a marked reduction inbacterial count, the rapid growth rates of bacteria make the presence ofeven a very few bacteria intolerable when sterile effluent is desired.

5 EXAMPLE 3 Filtering grade sand, about 0.5 mm. diameter, was soaked for12 hours in an aqueous 0.5 weight percent PE-I solution to establishequilibrium. All excess PEI was then washed oif with deionized water andan 18 inch high column of this treated sand was placed in a 36 inchvertical glass tube having a diameter of 1 inch. All equipment wassterilized by autoclaving prior to addition of the coated sand. The sandwas fluidized by a back wash with sterile water and then allowed tosettle with an inch of water above the sand level. The feed consisted of7 mls. of sterile water inoculated with Escherichia coli and wasfiltered at a gravity rate of 30 ml. per minute. Serial dilutions of theprefiltr-ate and post filtrate were employed to determine totalbacterial population. Untreated sand and sand coated with aluminumhydroxide were employed in otherwise identical runs for the purpose ofcomparison. The aluminum hydroxide coated sand was prepared by themethod described in U.S. Patent 2,832,473. Table III, below, is acompilation of the results obtained.

Once again the significance of the results resides in the necessity forcomplete removal of bacteria since even the relatively small numbersallowed to pass through into the filtrate by the aluminum hydroxidecoated sand are capable of rapid growth and rebuild-up of the bacterialpopulation.

Total removal of intestinal parasites by filtration through 0.6 mm;sand, coated using the same technique as described above, at rapidfiltration rates was also accomplished. Endoameba hisloylzica, Endolimaxhim and Enierobius vermicularia were used to inoculate a sterile waterinfiuent. Total removal of these microorganisms was observed at a flowrate of 2 gallons per minute per square foot of filter area.

EXAMPLE 4 The removal of microorganisms from a variety of liquids wasdemonstrated with DE having an 0.2 weight percent PEI coating employedas the filter media. Beer containing 400 microorganisms per ml. wasfiltered at a rate of a liter/min. through a No. 2 Buechner funnelcontaining a A inch thick layer of the coated filter aid with completeremoval of the microorganisms being observed. In a similar manner,kerosene contaminated by 130,000 microorganisms/ ml. was sterilized bypassage through this coated filter aid medium with no blinding orplugging of the filter media.

EXAMPLE 5 Extended filtration runs were conducted using an 0.25 sq. ft.pressure diatomaceous earth filter system with a flow rate of 4 gallonsper min/sq. ft. filter area. The DE was spray treated with an aqueousPEI solution to provide 40 grams of a coated filter aid containing 0.2weight percent PEI. A 600 gallon tank of water was seeded with 24 hourbroth cultures of Escherichia coli. Table IV, below, shows the bacterialcount of the pre-filtrate and postfiltrate at various time intervalsover a total period of almost 20 hours.

Table IV Bacterial Count/ml. Time Tank (influent) EXAMPLE 6 Thefollowing experiment was conducted to demonstrate the eifectiveness ofthe present method to remove viral organisms from fluids. In a series ofruns, 10.0 gram quantities of coated diatomaceous earth, having 0.2weight percent PEI was slurried with ml. of a standard nutrient mediumcontaining 0, 5, 10, or 20 weight per cent horse serum. Each such slurrywas filtered through a filter paper in a Beuchner funnel to form afilter bed and portions of the filtrate were tested in establishedmonolayer tissue cultures of bovine kidney tissue. No evidence oftoxicity of such filtrates to the tissue cultures was observed.Immediately after filtering the nutrient medium to form the filter bed,100 ml. of a suspension of infectious bovine rhinotracheitis virus,hereafter IBR, was passed through the filter bed. Serial dilutions ofthe filtrate from each filter bed were inoculated into tubes containingcultures of bovine kidney established cells. The cultures were thenincubated under good growing conditions and observed for the developmentof the characteristic cytopathological eifect indicative of the presenceof IBR virus. The suspension of IBR virus employed as starting materialrepresented harvests from tissue cultures of the virus which had beenpooled to provide 500 ml. of a suspension which assayed 10 tissueculture infectious doses-50 percent, hereafter TCID of IBR virus per 0.2ml. This value is determined from that dilution at which there is acytopathogenic effect observed in 50 percent of the culture tubesinoculated with said dilution. The results of these experiments showedno cytopathogenic efiect when corresponding assays were carried out withthe filtrates from the coated filter aid beds which had been preparedfrom slurries containing 0, 5, 10 and 20 weight percent horse serum.

EXAMPLE 7 The effectiveness of the method of the present invention inremoving microorganisms from commercially employed dry cleaning solventswas tested by inoculating a 1:1 weight ratio, tetrachloroethylene:watersolution, with various bacteria and then filtering the contaminatedsolution through forty grams of DE having an 0.2 weight percent coat ofPEI. A standard No. 2 Buechner funnel was employed. The dilution of thedry cleaning solvent was necessary since 100% tetrachloroethylene istoxic to the bacteria employed. The coated DE was prepared byspray-treatment of DE with an aqueous solution of PEI. Thetetrachloroethylene-water suspensions of the test organism were filteredand the number of bacteria present before and after filtration wereenumerated by standard plate counts. After 24 hours incubation at 37 C.counts of bacteria were obtained as listed in Table V, below, in numberof bacteria/ ml.

Table V Bacteria/ml.

CC] CCl2]IzO Untreated DE IEI coated DE Suspension Ire-filtra-Post-filtralrc-liltra- Post-filtration tion tion tion A. aerogcnrs16,000 8, 000 14, 000 S. aureus 17, 000 3,000 8,000 0 B. subtilis 19,000 2, 000 20, 000 0 EXAMPLE 8 The capacity of PEI treated DE to removemicroorganisms from air by filtration was demonstrated by placing a dryfilter medium, consisting of DE spray-treated with PEI to prepare acoated filter aid having 0.2 Weight percent PEI, in a No. 2 Buechnerfunnel with a No. 2 Whatman filter paper used to support the coatedfilter aid. This filter was then connected by means of a cored rubberstopper to a nitro-cellulose ester membrane filter. The system wasbrought to vacuum and the flow rate controlled at 600 liters/hr. Uponcompletion of a filtration run, the membrane filter (0.45 micron poresize) was cultured on Standard Methods Agar and the microorganismsenumerated. The results in total bacteria and fungi present at varioustime intervals is listed in Table VI, below:

EXAMPLE 9 The following experiment was conducted to determine theeflectiveness of the filter aid coating in preventing the growth ofmicroorganisms through the filter bed.

Diatomaceous earth was slurried with an 0.2 weight percent aqueoussolution of the polyalkylenepolyamine of Example 1, having an averagemolecular weight of 400, and formed into an approximately inch thickfilter bed in a standard Buechner funnel. The funnel was connected to a500 ml. filter flask containing 100 ml. of sterile nutrient broth andsuccessive portions of a heavily bacteria-contaminated water werefiltered through the coated filter medium over a three day period. Anyturbidity occurring in the nutrient broth was indicative of bacterialgrowth, in this case due to bacterial grow Time (hrs) Untreated DECoated DE Visible growth N 0 growth. Do.

Do. Do.

We claim: 1. A method for the removal of microorganisms from fluidswhich comprises passing a fluid containing microorganisms through ananionic-type filter aid having an adherent coating of an organic,cationic, polyelectrolyte, which has a minimum of about 10 cationiccharge sites per molecule, said coating constituting from about 0.01 to2 percent of the total Weight of the coated filter aid.

2. The method of claim 1 wherein said adherent cat-ionic coating is apolyethylenimine.

3. The method of claim 1 wherein said adherent cationic coating is apolyalkylenepolyamine.

4. A rapid-flow method for the removal of bacterial, fungal and viralmicroorganisms from fluids which comprises passing a fluid containing atleast one of said microorganisms through an anionic-type filter aidhaving an adherent coating of a predominantly linear, organic, cationic,polyelectrolyte which has a minimum of about 10 ca;ionic charge sitesper molecule; said coating constituting from about 0.05 to 0.2 weightpercent of the coated filter-aid.

5. The method of claim 1 wherein said microorganismcontaining fluid is aliquid.

6. The method of claim 1 wherein said microorganismcontaining fluid is agas.

7. The method of claim 5 wherein said liquid is water.

8. The method of claim 6 wherein said gas is air.

9. The method of claim 1 wherein said coated fil1er aid is periodicallyre-coated by adding an organic, cationic, polyelectrolyte, which has aminimum of about 10 cationic charge sites per molecule, to the influentfluid.

References Cited by the Examiner UNITED STATES PATENTS 2,040,818 4/1936Badollet 2l0506 2,247,711 7/ 1941 Ralston 21064 2,692,231 10/1954Stayner et al 210--64 2,732,350 1/1956 Clarke 2101 XR 2,841,526 7/1958Gustus 16772 MORRIS O. WOLK, Primary Examiner.

1. A METHOD FOR THE REMOVAL OF MICROORGANISMS FROM FLUIDS WHICHCOMPRISES PASSING A FLUID CONTAINING MICROORGANISMS THROUGH ANANIONIC-TYPE FILTER AID HAVING AN ADHERENT COATING OF AN ORGANIC,CATIONIC, POLYELECTROLYTE, WHICH HAS A MINIMUM OF ABOUT 10 CATIONICCHARGE SITES PER MOLECULE, SAID COATING CONSTITUTING FROM ABOUT 0.01 TO2 PERCENT OF THE TOTAL WEIGHT OF THE COATED FILTER AID.